Rhamnosyl-tranferase gene and uses thereof

ABSTRACT

An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having a flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase catalytic activity and its uses.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a nucleic acid encoding a1-2-rhamnosyl-transferase and uses thereof. More particularly, thepresent invention relates to a multistep process of convertinghesperidin from orange peels to the sweetener neohesperidindihydrochalcone (NHDC), in which, in one of the steps,1-2-rhamnosyl-transferase in the presence of activated rhamnose is usedin a rhamnosylation reaction to convert hesperidinase-treated hesperidin(H7G) to neohesperidin. Further particularly, the present inventionrelates to genetically modified plants of the Citrus genus including anantisense or sense (for co-suppression) construct which comprises theabove nucleic acid or a gene knock-out integrated construct to provideless bitter grapefruits, pomelos and other citrus containing bitterflavanoid glycosides.

The bitter flavanones naringin and neohesperidin (FIG. 1) are producedonly in young leaves and fruits of a few citrus species, such asgrapefruit and pomelo, accumulate in a brief few week period, and remainthrough maturity (Castillo et al., 1992). Isomerically structured, yettasteless flavanones, such as hesperidin (FIG. 1) are produced inoranges at the same time in development (Castillo et al., 1993). Thedifferences between the tastelessness of orange hesperidin and thebitterness of grapefruit and pomelo naringin and neohesperidin depend ona specific set of glycosylation reactions (glucosylation andrhamnosylation) as further detailed hereinunder.

Low consumption of grapefruit is due in part to the bitter flavor of itsjuice and flesh (Matthews et al., 1990). This bitterness is due to thepresence of large amounts of the flavanone glycoside naringin, as wellas limonin in the juice. While limonin is a problem limited to the juice(Guadagni et al., 1973), in the intact fruit it appears in the tastelessA ring monolactone and forms the bitter dilactone only after macerationin the acidic juice environment (Matthews et al., 1990).

Thus, bitterness in the fruit can be decreased by reducing naringinlevels. The bitterness in commercially prepared grapefruit juice ispresently diminished to levels more acceptable by consumers by usingresins that adsorb some bitter compounds or by treating the juice withthe immobilized enzyme naringinase (Jimeno et al., 1987; Nikdel et al.,1989).

Commercial naringinase preparations typically consist of two enzymes,mainly α-rhamnosidase and some β-glucosidase, which successivelyhydrolyze one or both the sugar groups from naringin, leaving thetasteless compound prunin (naringenin-7-O-glucoside). Removal of theterminal rhamnose removes about 95% of the bitterness. Evidently, thisprocedure does not solve the problem of bitterness of whole fruit orhome prepared juice.

A better approach which also addresses the above problem would,therefore, be to regulate the levels of naringin within the plantitself.

However, because of high heterozygousity of the commercial citrusvarieties, classical plant breeding programs, will be hard put to yieldidentical varieties, yet having significantly less bitter fruit byreducing naringin. It will be appreciated in this respect that thepresently available low naringin varieties (such as the Texas Red) arealso low yielding.

While reducing the present invention to practice, studies of thenaringin flavonoid metabolism (see below) assisted (i) in developingnovel approaches to the problem; (ii) to further study the criticalglycosylation steps that produce naringin; and (iii) in using thisknowledge to modulate the degree of bitterness.

Citrus flavonoids ubiquity and biosynthesis: The genus Citrus containsmany flavonoid glycosides that differ either in the structure of theaglycone or their sugar moieties. The major flavonoid in pomelo andgrapefruit peel is naringin, while sweet orange peel contains hesperidin(Horowitz and Gentili, 1977 and FIG. 1). Peels of sour orange,trifoliate orange and Ponderosa lemon contain neohesperidin (FIG. 1).Neohesperidin and naringin are flavanone glycosides that contain thesame disaccharide, β-neohesperidose (2-O-α-L-rhamnosyl-β-D-glucose),which is attached at position C-7 of different aglycones, hesperetin andnaringenin, respectively. Hesperidin and narirutin are isomers,respectively, of the above compounds that contain the disacchariderutinose (6-O-α-L-rhamnosyl-β-D-glucose).

These flavonoids have some remarkable taste properties. Naringin andneohesperidin are extremely bitter, while narirutin and hesperidin arenearly tasteless (Horowitz and Gentili, 1986; Naim et al., 1986).

Flavonoids in Citrus: The highest naringin levels are associated withvery young developing leaves and fruit tissue. Undifferentiated cells ofCitrus paradisi (grapefruit) were able to biotransform exogenousnaringenin and hesperetin, into prunin (naringenin-7-O-glucoside) andhesperetin-7-O-glucose (H-7-G), respectively. Further 1-2-rhamnosylafionresulting in naringin or neohesperidin synthesis was not detected,although 1-6-rhamnosylation resulting in narirutin was observed(Lewinsohn et al., 1986, 1989b).

All of the above suggests that the blockage in the stepwise productionof the naringenin from prunin (naringenin-7-O-glucoside) in theundifferentiated Citrus cells is caused by the absence of a specificrhamnosyl transferase activity.

Lewinsohn et al. detected chalcone-synthase and UDP-glucose,flavanone-7-O-glucosyl-transferase activities in cell-free extracts ofCitrus (Lewinsohn et al. 1989a). Partial purification of the glucosyltransferase has been recently reported and some of its characteristicsare therefore known (McIntosh and Mansell, 1989).

The glucosylated flavanone was further rhamnosylated. Chalcone-synthaseactivity was detected in cell-free extracts derived from young leavesand fruits.

Young fruits (2 millimeter diameter) had the highest chalcone synthaseactivity. In earlier studies it was shown that the glycosylation of theaglycone, naringenin, in undifferentiated Citrus cells occurs in twosteps. First there was an initial glucosylation resulting in prunin(Lewinsohn et al. 1986, 1989b), and then a further rhamnosylation ofprunin occurred when exogenous UDP-glucose and NADPH were added to theextract forming the end-product naringin (Lewinsohn et al 1989b).

Prunin was also shown to be a likely intermediate in the biosynthesis ofnaringin in immature grapefruit fruits (Berhow and Vandercook 1989).

Several glucosyl-transferases from plants catalyze the transfer of thesugar moiety from an activated UDP-sugar to a specific site on theflavonoids (Hahlbrock, 1981). Enzymatic preparations from plantscatalyze the conversion of UDP-glucose to UDP-rhamnose in the presenceof NADPH (Barber, 1962). The conversion is due to at least threedifferent enzymatic activities requiring NADH or NADPH. It was notpreviously known how this conversion is catalyzed in Citrus. A coupledassay using UDP-glucose with NADPH was developed, which formed arhamnosylated flavanone glycoside, indicative of aUDP-rhamnose:flavanone-7-O-glucoside-2′-O-rhamnosyl-transferase activity(α-1-2 rhamnosyl transferase). A system was thereafter developed todirectly biosynthesize radiolabeled UDP-rhamnose from [¹⁴C]-UDP glucoseand NADPH for use in direct assay of the α-1-2 rhamnosyl transferaseactivity during purification of the α-1-2 rhamnosyl transferase. Thedirect assay facilitated the purification of the α-1-2 rhamnosyltransferase. Once both rhamnosyl transferase substrates were availableit was possible to purify the enzyme. Purity to homogeneity of the α-1-2rhamnosyl transferase in a four step procedure, which includes S-200 gelfiltration, affinity column, ion exchange-FPLC and reverse-phase HPLC(Bar-Peled et al. 1991) was achieved and an antibody to it was elicited.These findings allowed to isolate the gene, which is the subject of thisinvention.

Semi-artificial sweetener: Neohesperidin and naringin from the peels ofsour oranges and grapefruits are being converted into neohesperidindihydrochalcone (NHDC), an intense sweetener used in the EU that is2,000 fold sweeter than sugar (see right path on FIG. 1). NHDC isespecially useful when long-lasting sweetness is desired, such as inchewing gum or in admixture with one of the artificial sweeteners.Currently, world-wide production of this NHDC is limited by the amountof neohesperidin and naringin available from these lesser grown Citrusspecies (less than 10% of total Citrus cultivated).

Unfortunately, the vast majority of citrus processed (oranges) containlarge amounts of the hesperidin, an isomer having the terminal rhamnoseattached by a 1→6 linkage instead of the 1→2 linkage. Hesperidin cannotbe converted to the sweetener and is currently a waste by-product.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, a nucleic acid encoding a1-2-rhamnosyl-transferase to effect a multistep process of convertinghesperidin from orange peels to the sweetener neohesperidindihydrochalcone (NHDC), and to provide genetically modified plants ofthe Citrus genus including an antisense or sense (for co-suppression)construct thereof, or knock-out integrated construct, to provide lessbitter grapefruits, pomelos and other citrus containing bitter flavanoidglycosides.

SUMMARY OF THE INVENTION

While reducing the present invention to practice, a novel rhamnosyltransferase gene from citrus responsible for producing the bitterflavanoids naringin and neohesperidin was isolated, sequenced, andcharacterized. This gene can be used in antisense or overproducing senseconstructs to decrease the bitterness in grapefruit, and in senseconstructs as part of the bioconversion of hesperidin from orange wasteproducts to neohesperidin, used for the production of neohesperidindihydrochalcone (NHDC), a sweetener. A knock-out construct can be usedto knock-out the endo rhamnosyl transferase gene.

Thus, according to one aspect of the present invention there is providedan isolated polynucleotide comprising a nucleotide sequence encoding apolypeptide having a flavanone-7-O-glucoside-2″-O-rhamnosyl-transferasecatalytic activity.

According to another aspect of the present invention there is provided acell genetically modified to include, in an expressible senseorientation, a nucleotide sequence encoding aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase.

According to still another aspect of the present invention there isprovided a plant cell of a plant species naturally expressing aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase messenger RNA,wherein the plant cell is genetically modified to include, in anexpressible antisense orientation, a nucleotide sequence encoding anantisense RNA molecule being capable of in vivo base pairing with theflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase messenger RNA, tothereby render the flavanone-7-O-glucoside-2″-O-rhamnosyl-transferasemessenger RNA, when expressed, amenable to degradation by nucleasespresent in the plant cell.

According to yet another aspect of the present invention there isprovided a transgenic plant of a species naturally expressing aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase messenger RNA,wherein the transgenic plant is genetically modified to include, in anexpressible antisense orientation, a nucleotide sequence encoding anantisense RNA molecule being capable of in vivo base pairing with theflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase messenger RNA, tothereby render the flavanone-7-O-glucoside-2″-O-rhamnosyl-transferasemessenger RNA, when expressed, amenable to degradation by nucleasespresent in the transgenic plant.

According to a further aspect of the present invention there is provideda plant cell of a plant species naturally expressing aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase, wherein the plantcell is genetically modified to include, in an overexpressible senseorientation, a nucleotide sequence encoding a sense RNA molecule beingcapable of inducing a co-suppression effect to thereby reduce productionof the flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in the plantcell.

According to yet a further aspect of the present invention there isprovided a transgenic plant of a species naturally expressing aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase, the plant isgenetically modified to include, in an overexpressible senseorientation, a nucleotide sequence encoding a sense RNA molecule beingcapable of inducing a co-suppression effect to thereby reduce productionof the flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in the plantcell.

According to still a further aspect of the present invention there isprovided a transgenic plant or cell of a plant species naturallyexpressing a flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase, thetransgenic plant or cell being genetically modified to knock-out a geneencoding said flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase tothereby abolish production of saidflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in the transgenicplant or cell.

According to an additional aspect of the present invention there isprovided a commercial process of producing neohesperidin comprising thesteps of: (a) extracting hesperidin from a hesperidin producing speciesof the genus Citrus; (b) treating the hesperidin with a hesperidinase,thereby obtaining hesperetin-7-glucoside; and (c) treating thehesperetin-7-glucoside with aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in the presence ofactivated rhamnose, thereby obtaining neohesperidin.

According to yet another additional aspect of the present inventionthere is provided a method of modifying a rhamnose-1-6-glucose linkageof a chemical compound to a rhamnose-1-2-glucose linkage comprising thesteps of: (a) enzymatically treating the chemical compound to remove arhamnose group thereof, thereby obtaining a first derivative of saidchemical compound having a terminal glucose group; and (b) enzymaticallytreating the first derivative with recombinantflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in the presence ofactivated rhamnose, thereby obtaining a second derivative chemicalcompound having the rhamnose-1-2-glucose linkage.

According to one further aspect of the present invention there isprovided a commercial process of producing neohesperidin dihydrochalconecomprising the steps of: (a) extracting hesperidin from a hesperidinproducing species of the genus Citrus; (b) treating the hesperidin witha hesperidinase, thereby obtaining hesperetin-7-glucoside; (c) treatingthe hesperetin-7-glucoside with aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in the presence ofactivated rhamnose, thereby obtaining neohesperidin; (d) treating theneohesperidin with an alkali, thereby obtaining neohesperidin chalcone;and (e) reducing the neohesperidin chalcone, thereby obtainingneohesperidin dihydrochalcone.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a nucleic acid encoding a1-2-rhamnosyl-transferase to effect a multistep process of convertinghesperidin from orange peels to the sweetener neohesperidindihydrochalcone (NHDC), and to provide genetically modified plants ofthe Citrus genus to provide less bitter grapefruits, pomelos and othercitrus containing bitter flavanoid glycosides.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings, wherein:

FIG. 1 depicts a prior art process (right path) and a process accordingto the present invention (left path) of producing the sweetener NHDC;and

FIG. 2 shows the sequence of the coding strand of a1-2-rhamnosyl-transferase cDNA according to the present invention, andits translation into a protein shown in single letter code.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a 1-2-rhamnosyl-transferase gene and therecombinant protein product hereof, which can be used in a multistepprocess of converting hesperidin from orange peels to the sweetenerneohesperidin dihydrochalcone (NHDC), and in providing geneticallymodified plants of the genus Citrus characterized by lowered bitterness.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting. Generally, thenomenclature used herein and the laboratory procedures in recombinantDNA technology described below are those well known and commonlyemployed in the art.

While reducing the present invention to practice, a polynucleotide wasisolated, having a nucleotide sequence encoding a polypeptide having aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase catalytic activity.The isolation of the flavanone-7-O-glucoside-2″-O-rhamnosyl-transferasegene was not at all trivial. Repeated attempts to isolate the gene viascreening of either conventional cDNA libraries or expression cDNAlibraries using oligonucleotides as well as antibodies raised againstpurified flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase failed toyield positive clones. The successful approach of isolating theflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase gene, which includedreverse transcriptase polymerase chain reaction (RT-PCR) usingdegenerate primers followed by rapid amplification of cDNA ends (RACE),is further described in the Examples section that follows.

Having the cDNA in hand, screening of genomic libraries can readily beperformed for isolating a genomic clone including a polynucleotidehaving introns and exons, the exons of which correspond to the cDNAsequence or parts thereof, and encode forflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase. Both thecomplementary and genomic polynucleotides according to the presentinvention can be used to direct the synthesis of a messenger RNA (mRNA)encoding flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase.

According to a preferred embodiment of the present invention, theisolated polynucleotide, either in its cDNA or genomic DNA form, whollyor partially synthetic (the term “isolated” may encompass all thesepossibilities) is ligated to a vector, preferably, an expression vector,either in sense or antisense orientation. The vector preferably selectedis propagatable in plant cells and/or in microorganism cells, such as inbacteria. The vector can be of a type capable of directing theintegration of sequences thereof into the genome of the recipient cell.Thus, within the cell, the nucleic acid may be incorporated, or not,within a chromosome. There may be more than one nucleotide sequence perhaploid genome. This, for example, enables increased expression. Avector comprising a polynucleotide according to the present inventionneed not include a promoter, particularly if the vector is to be used tointroduce the nucleic acid into cells for recombination into the genome.

However, as further detailed hereinunder, a promoter is preferablyincluded in the vector.

The present invention also encompasses the expression product of any ofthe nucleic acid sequences disclosed and methods of making theexpression product by expression from encoding nucleic acid thereforunder suitable conditions in suitable host cells. Those skilled in theart are well able to construct vectors and design protocols forexpression and recovery of products of recombinant gene expression.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al. 1989, Cold Spring Harbor Laboratory Press. Transformationprocedures depend on the host used, but are well known. Many knowntechniques and protocols for manipulation of nucleic acids, for example,in preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins are described in detail in Short Protocols in MolecularBiology, Second Edition, Ausubel et al. ed., John Wiley & Sons, 1992.The disclosures of Sambrook et al. and Ausubel et al. are incorporatedherein by reference.

According to one embodiment of the present invention the nucleotidesequence is as set forth in SEQ ID NO:20 or a functional part thereof.By functional part it is meant a part capable of directing the synthesisof a polypeptide having aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase catalytic activity.

According to another embodiment of the present invention the nucleotidesequence shares between 50 and 100%, preferably between 60 and 100%,more preferably between 70 and 100%, yet more preferably between 80 and100%, still more preferably between 90 and 100% identical bases with SEQID NO:20 or a functional part thereof, as determined using a sequenceanalysis software package developed by the Genetic Computer Group (GCG)at the University of Wisconsin with gap creation penalty of 50 and gapextension penalty of 3, provided that the nucleotide sequence encodes apolypeptide having a flavanone-7-O-glucoside-2″-O-rhamnosyl-transferasecatalytic activity.

According to yet another preferred embodiment of the present invention,the nucleotide sequence is hybridizable with SEQ ID NO:20 or afunctional part thereof under stringent hybridization, moderatehybridization or mild hybridization, wherein stringent hybridization iseffected by a hybridization solution of 6×SSC and 1% SDS, hybridizationtemperature of 65° C., final wash solution of 0.1×SSC and final wash at60° C., moderate hybridization is effected by a hybridization solutionof 6×SSC and 1% SDS, hybridization temperature of 58° C., final washsolution of 0.5×SSC and final wash at 50° C., whereas mild hybridizationis effected by a hybridization solution of 6×SSC and 1% SDS,hybridization temperature of 50° C., final wash solution of 2×SSC andfinal wash at 40° C. Thus, a hybridization according to the presentinvention is effected by a hybridization solution of 6×SSC and 1% SDS,hybridization temperature of 50-65° C., final wash solution of0.1-2.0×SSC and final wash at 40-60° C.

According to a preferred embodiment of the present invention, thenucleotide sequence encodes a polypeptide as set forth in SEQ ID NO:21or a functional part thereof. However, the polypeptide encoded by thenucleotide sequence of the present invention can share between 20 and100%, preferably between 60 and 100%, more preferably between 70 and100%, yet more preferably between 80 and 100%, still more preferablybetween 90 and 100% identical or conserved amino acids with SEQ ID NO:21or a functional part thereof, as determined using a sequence analysissoftware package developed by the Genetic Computer Group (GCG) at theUniversity of Wisconsin with gap creation penalty of 12 and gapextension penalty of 4, provided that the polypeptide has aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase catalytic activity.

A polynucleotide according to various aspects of the present inventionmay have the sequence of aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase gene or be a mutant,variant, derivative or allele of the sequence provided. Preferredmutants, variants, derivatives and alleles are those which encode aproduct (RNA or polypeptide) which retains a functional characteristicof the product encoded by the wild-type gene. Changes to a sequence, toproduce a mutant, variant or derivative, may be by one or more ofaddition, insertion, deletion or substitution of one or more amino acidsin an encoded polypeptide product. Of course, changes to the nucleicacid which make no difference to the encoded amino acid sequence areincluded.

In a preferred embodiment of the present invention a polynucleotidemolecule comprises a nucleotide sequence which encodes an amino acidsequence shown in SEQ ID NO:21. The nucleotide sequence may comprise anencoding sequence shown in SEQ ID NO:20 or may be a mutant, variant,derivative or allele thereof encoding the same amino acid sequence, or asequence which retains a functional characteristic of the productencoded by the wild-type gene.

Sequences comprising changes to or differences from the sequences shownmay also be employed in the present invention, as discussed herein.

According to a preferred embodiment of the present invention thenucleotide sequence originates from a species of the genus Citrus, suchas grapefruit and pomelo.

The nucleotide sequence information provided herein or any part thereofmay be used in a data-base search to find homologous sequences,expression products of which can be tested forflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase activity.

A further aspect of the present invention provides a method ofidentifying and cloning aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase gene homolog from aplant species other than pomelo which method employs a nucleotidesequence derived from SEQ ID NO:20. Nucleic acid libraries may bescreened using techniques well known to those skilled in the art andhomologous sequences thereby identified then tested. Hybridization mayinvolve probing nucleic acid and identifying positive hybridizationunder suitable stringent conditions (in accordance with knowntechniques) and/or use of oligonucleotides as primers in a method ofnucleic acid amplification, such as PCR. For probing, preferredconditions are those which are stringent enough for there to be a simplepattern with a small number of hybridizations identified as positivewhich can be investigated further. It is well known in the art toincrease stringency of hybridization gradually until only a few positiveclones remain. As an alternative to probing, though still employingnucleic acid hybridization, oligonucleotides designed to amplify DNAsequences may be used in PCR reactions or other methods involvingamplification of nucleic acid, using routine procedures. See forinstance “PCR protocols; A Guide to Methods and Applications”, Eds.Innis et al. 1990, Academic Press, New York. Assessment of whether ornot such a PCR product corresponds to aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase may be conducted invarious ways. A PCR band from such a reaction might contain a complexmix of products. Individual products may be cloned and each oneindividual product may be cloned and each one individually screened. Itmay be analyzed by transformation to assess function on introductioninto a plant of interest.

Thus, included within the scope of the present invention arepolynucleotide molecules which encode amino acid sequences which arehomologs of flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase ofpomelo. Homology may be at the nucleotide sequence and/or amino acidsequence level. Preferably, the nucleic acid or amino acid sequence of ahomolog, or a mutant, allele, derivative or variant (see above) shareshomology with SEQ ID NOs:20 or 21, respectively, preferably at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 75.%, or at least about 80% homology, most preferably at leastabout 90% homology, and the encoded product shares a phenotype with theflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase gene. “Homology” maybe understood to refer to similarity, in functional terms, in an aminoacid sequence, as is standard in the art. Thus, for example, a %similarity figure will include amino acid differences that have littleor no functional significance, such as leucine to isoleucine. Otherwise,homology may be taken to refer to identity. These definitions arefurther elaborated hereinabove. Thus, for example, gene homologs fromother members of the genus Citrus may be identified.

In certain embodiments, an allele, variant, derivative, mutant orhomolog of the specific sequence may show little overall homology, sayabout 20% or about 25%, or about 30%, or about 35% or about 40% or about45%, with the specific sequence. However, in functionally significantdomains or regions the amino acid homology may be much higher. Deletionmutagenesis, for example, may be used to test the function of a regionof the polypeptide and its role in or necessity for catalytic activity.

Also according to the invention there is provided a plant cell havingincorporated into its genome a sequence of nucleotides as provided bythe present invention, under operative control of a promoter for controlof expression of the encoded polypeptide. A further aspect of thepresent invention provides a method of making such a plant cellinvolving introduction of a vector comprising the sequence ofnucleotides into a plant cell and causing or allowing recombinationbetween the vector and the plant cell genome to introduce the sequenceof nucleotides into the genome.

The present invention further encompasses a plant comprising a plantcell comprising nucleic acid according to the present invention e.g., asa result of introduction of the nucleic acid into the cell or anancestor thereof, and selfed or hybrid progeny and any descendant ofsuch a plant, also any part or propagule of such a plant, progeny ordescendant, including sexually or apomictically obtained seed andvegetatively propagated plant material.

The principal characteristics which may be altered using the presentinvention are controlling the bitterness of fruits of plants of thegenus Citrus as further detailed hereinunder. Over-expression of thegene product of the flavanone-7-O-glucoside-2″-O-rhamnosyl-transferasegene may lead to either bitterer fruits or less bitter fruits via aco-suppression mechanism; under-expression may lead to less bitterfruits. Down-regulation may be achieved, for example, with “genesilencing” techniques such as anti-sense or sense regulation, discussedfurther below. It can also be achieved with gene knockout.

This degree of control is useful to provide fruits of desired bitternesswhich can be used in the fresh fruit market or in the juice productionindustry, diminishing the need for de-bittering processes.

In preparing a vector according to the present invention various DNAfragments may be manipulated as necessary to create the desired vectoror construct. This includes using linkers or adaptors as necessary toform suitable restriction sites or to eliminate unwanted restrictionsites or other like manipulations which are known to those of ordinaryskill in the art.

Promoters which are known or found to cause transcription of a selectedgene in plant cells can be used in the present invention. Such promotersmay be obtained from plants, plant pathogenic bacteria or plant viruses,and include, but are not necessarily limited to, the 35S and 19Spromoters of cauliflower mosaic virus (CaMV35S and CaMV19S), thefull-length transcript promoter from the figwort mosaic virus (FMV35S)and promoters isolated from plant genes such as EPSP synthase andssRUBISCO genes, and promoters obtained from T-DNA genes ofAgrobacterium tumefaciens, such as nopaline and mannopine synthases. Theparticular promoter selected should be capable of causing sufficientexpression of sense or antisense RNA.

Particularly useful promoters for use in the present invention are fruitspecific promoters which are expressed, for example, during ethyleneproduction in the fruit and the full-length transcript promoter from thefigwort mosaic virus (FMV35S). The FMV35S promoter is particularlyuseful because of its ability to cause uniform and high levels ofexpression.

The DNA sequence of a FMV35S promoter is presented in U.S. Pat. No.5,512,466 and is identified as SEQ ID NO:17 therein. Examples of fruitspecific promoters include the E8, E4, E17 and J49 promoters from tomato(Lincoln, J. and Fischer, R. (1988). Diverse mechanisms for theregulation of ethylene-inducible gene expression. Mol Gen Genet 212,71-75), as well as the 2A11 promoter as described in U.S. Pat. No.4,943,674.

As used herein, the term “CaMV35S” or “FMV35S” promoter includevariations of these promoters, e.g., promoters derived by means ofligation with operator regions, random or controlled mutagenesis,addition or duplication of enhancer sequences, etc.

A 3′ non-translated region is preferably included in a sense vectoraccording to the present invention and includes a polyadenylation signalwhich functions in plants to cause the addition of polyadenylatednucleotides to the 3′ end of a sense RNA sequence. Examples of suitable3′ regions are the 3′ transcribed, non-translated regions containing thepolyadenylation signal of the tumor-inducing (Ti) plasmid genes ofAgrobacterium, such as the nopaline synthase (NOS) gene, and plant geneslike the 7S soybean storage protein genes and the pea E9 small subunitof the RuBP carboxylase gene (ssRUBISCO).

A sense RNA produced by a vector of the present invention alsopreferably contains a 5′ non-translated leader sequence. This sequencecan be derived from the promoter selected to express the gene, and canbe specifically modified so as to increase translation of the sense RNA.The 5′ non-translated regions can also be obtained from viral RNA's,from suitable eukaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to vector constructs wherein thenon-translated region is derived from the 5′ non-translated sequencethat accompanies the promoter sequence. Rather, the non-translatedleader sequences can be part of the 5′ end of the non-translated regionof the native coding sequence, or part of the promoter sequence, or canbe derived from an unrelated promoter or coding sequence.

In a preferred embodiment according to the present invention, the vectorthat is used to introduce the sense or antisense RNA into the host cellsor plants will comprise an appropriate selectable marker. In a morepreferred embodiment according to the present invention the vector is aplant expression vector comprising both a selectable marker and anorigin of replication. In another most preferred embodiment according tothe present invention the vector will be a shuttle vector, which canpropagate both in E. coli (wherein the construct comprises anappropriate selectable marker and origin of replication) and becompatible for propagation or integration in the genome of the organismof choice. In yet another embodiment, the construct comprising thepromoter of choice and the gene of interest is placed in a viral vectorwhich is used to infect the cells. This virus may be integrated in thegenome of the organism of choice or may remain non-integrated.

The promoter of choice that is used in conjunction with this inventionwill comprise any suitable promoter as further detailed herein. It willbe appreciated by one skilled in the art, however, that it is necessaryto make sure that the transcription start site(s) will be locatedupstream of the open reading frame. In a preferred embodiment of thepresent invention, the promoter that is selected will comprise anelement that is active in the particular host plant cells of interest.

These elements may be selected from transcriptional regulators thatactivate the transcription of genes essential for the survival of thesecells in conditions of stress or starvation, including the heat shockproteins. Promoters containing this type of sequence may advantageouslybe used according to the present invention.

As further detailed hereinabove, DNA sequences encoding thetranslational start site (ATG) of the gene to be expressed, will beplaced downstream of the transcription start site(s). Any equivalentfunctional element selected from similar elements in this or otherorganisms may be used as appropriate in the organism of choice.Equivalent functional elements will include elements with syntheticbases, or elements found in other genes of plants as well as elementsfound in genes of other unicellular or multicellular organisms.

According to one embodiment of the present invention secretion of theprotein out of the cell is preferred. In this embodiment the constructwill comprise a signal sequence to effect secretion as is known in theart. A signal sequence that is recognized in the active growth phasewill be most preferred. As will be recognized by the skilled artisan,the appropriate signal sequence should be placed immediately downstreamof the translational start site (ATG), and in frame with the codingsequence of the gene to be expressed.

Introduction of the construct or vector into the cells is accomplishedby any conventional method for transfection, infection or the like, asis known in the art. In constructs comprising a selectable marker thecells may be selected from those bearing functional copies of theconstruct. If the plasmid comprising the gene of interest is episomalthe appropriate selective conditions will be used during growth. Stabletransfectants and stable cell lines may be derived from the transfectedcells in appropriate cases, in order to conveniently maintain thegenotype of interest. Cell growth is accomplished in accordance with thecell type, using any standard growth conditions as may be suitable tosupport the growth of the specific cell line.

A DNA construct of the present invention can be inserted into the genomeof a plant by any suitable method. Suitable plant transformation vectorsinclude those derived from a Ti plasmid of Agrobacterium tumefaciens,such as those disclosed by Bevan and Chilton, Ann. Rev. Genetics 16,357-384, 1982; U.S. Pat. No. 4,940,838 and others. In addition to planttransformation vectors derived from the Ti or root-inducing (R1)plasmids of Agrobacterium, alternative methods can be used to insert theDNA constructs of this invention into plant cells. Such methods mayinvolve, for example, the use of liposomes, electroporation, chemicalsthat increase free DNA uptake, particle gun technology, andtransformation using viruses.

The construction of vectors capable of being inserted into a plantgenome via Agrobacterium tumefaciens mediated delivery is known to thoseof ordinary skill in the art. Typical plant cloning vectors compriseselectable and scoreable marker genes, T-DNA borders, cloning sites,appropriate bacterial genes to facilitate identification oftransconjugates, broad host-range replication and mobilization functionsand other elements as desired.

If Agrobacterium mediated delivery is chosen, once the vector has beenintroduced into the disarmed Agrobacterium strain, the desired plant canthen be transformed. Any known method of transformation that will workwith the desired plant can be utilized.

Thus, further according to the present invention there are provided aplant cell and a transgenic plant of a species naturally expressing aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase messenger RNA, suchas, but not limited to, grapefruit or pomelo, wherein the plant cell ortransgenic plant are genetically modified to include, in an expressibleantisense orientation, a nucleotide sequence encoding an antisense RNAmolecule being capable of in vivo base pairing with theflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase messenger RNA, tothereby render the flavanone-7-O-glucoside-2″-O-rhamnosyl-transferasemessenger RNA, when expressed, amenable to degradation by nucleasespresent in the plant cell or transgenic plant. The plant cell ortransgenic plant according to this aspect of the present inventionexhibit either very low or noflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase activity.

It will be appreciated that the nucleotide sequence encoding anantisense RNA molecule being capable of in vivo base pairing with theflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase messenger RNAaccording to the present invention can be either extrachromosomal orintrachromosomal. In any case, as further detailed herein, thenucleotide sequence is preferably ligated to an expression vector in theantisense orientation downstream to a promoter element and optionallyother gene expression control elements.

The nucleotide sequence encoding an antisense RNA molecule being capableof in vivo base pairing with theflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase messenger RNAaccording to the present invention can be as set forth in SEQ ID NO:20or a portion thereof longer than 10 nucleotides, preferably longer than15 nucleotides, more preferably longer than 20 nucleotides, mostpreferably longer than 30 or 40 nucleotides, so as to ensure specificbase pairing with the endogenousflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase messenger RNAproduced by the plant cell under physiological conditions.

Alternatively, the nucleotide sequence encoding an antisense. RNAmolecule being capable of in vivo base pairing with theflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase messenger RNAaccording to the present invention can share between 80 and 100%identical bases with SEQ ID NO:20 or a portion thereof longer than 10nucleotides, preferably longer than 15 nucleotides, more preferablylonger than 20 nucleotides, most preferably longer than 30 or 40nucleotides, as determined using a sequence analysis software packagedeveloped by the Genetic Computer Group (GCG) at the University ofWisconsin with gap creation penalty of 50 and gap extension penalty of3, so as to ensure specific base pairing with the endogenousflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase messenger RNAproduced by the plant cell.

Still alternatively, the nucleotide sequence encoding an antisense RNAmolecule being capable of in vivo base pairing with theflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase messenger RNAaccording to the present invention can be hybridizable in vitro with SEQID NO:20 or a portion thereof longer than 10 nucleotides, preferablylonger than 15 nucleotides, more preferably longer than 20 nucleotides,most preferably longer than 30 or 40 nucleotides, under hybridizationselected from the group consisting of stringent hybridization, moderatehybridization and mild hybridization, wherein stringent hybridization iseffected by a hybridization solution of 6×SSC and 1% SDS, hybridizationtemperature of 65° C., final wash solution of 0.1×SSC and final wash at60° C., moderate hybridization is effected by a hybridization solutionof 6×SSC and 1% SDS, hybridization temperature of 58° C., final washsolution of 0.5×SSC and final wash at 50° C., whereas mild hybridizationis effected by a hybridization solution of 6×SSC and 1% SDS,hybridization temperature of 50° C., final wash solution of 2×SSC andfinal wash at 40° C., so as to ensure specific base pairing with theendogenous flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase messengerRNA produced by the plant cell. These alternative hybridizationconditions are useful in evaluating hybridization capabilities ofsequences according to all of the aspects of the present invention.Other hybridization conditions can also be employed. Step by stepprotocols for the above specified hybridizations and the mentioned otherhybridizations are found in Molecular Cloning: a Laboratory Manual: 2ndedition, Sambrook et al. 1989, Cold Spring Harbor Laboratory Press,which is incorporated by reference as if fully set forth herein.

Further according to the present invention there is provided a cellgenetically modified to include, in an expressible sense orientation, anucleotide sequence encoding aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase. The nucleotidesequence according to this aspect of the present invention can becomplementary DNA or genomic DNA, and it can be either extrachromosomalor intrachromosomal. It is preferably ligated to an expression vector inthe sense orientation and has any of the features described herein abovewith respect to the isolated polynucleotide according to the presentinvention. The cell according to this aspect of the present inventioncan be either a microorganism (either a prokaryote or a eukaryote) or acell of a multicellular organism, such as a cell of a multicellularplant. In any case, for reasons further detailed hereinunder, theselected cell is preferably capable of producing an activated rhamnose,either in the form of UDP-rhamnose or of dTDP-rhamnose.

Suitable microorganism which are known to produce dTDP-rhamnose include,but are not limited to, Lactobacillus Spp., such as Lactobacillus caseiiand Lactobacillus delbrueckeii and yeast such as Saccharomycescerevisiae.

Cells of suitable multicellular plants include, for example, cells ofplants of the genus Citrus, such as, but not limited to, grapefruit andpomelo, both being capable of producing UDP-rhamnose. Additionalexamples include cells of tobacco (Nicotiana tabacum), grapes (Vitisviniferis) and carrot (Daucus carota).

Further according to the present invention there is provided a plantcell or a transgenic plant of a plant species naturally expressing aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase, such as, but notlimited to, grapefruit and pomelo, the plant cell or transgenic plantbeing genetically modified to include, in an overexpressible senseorientation, a nucleotide sequence encoding a sense RNA molecule beingcapable of inducing a co-suppression effect to thereby reduce productionof the flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in the plantcell or the transgenic plant.

The transgenic plants according to the present invention, eitherexpressing the nucleotide sequence according to the present invention asherein described in an antisense form or in an overexpressible senseform, both are characterized by reduced production offlavanone-7-O-glucoside-2″-O-rhamnosyl-transferase. Plants can thereforebe selected via taste tests to produce fruits of desired degree ofbitterness, to provide, for example, less bitter fruits. Varieties withoptimal perceived bitterness levels can be propagated vegetatively byland grafting or other procedures known to the ordinary artisan, and canbe used to provide fruits to the fresh fruit market and to producejuices, such as grapefruit juice, that do not require de-bittering.

Furthermore, according to still another aspect of the present inventionthere is provided a transgenic plant or cell of a plant speciesnaturally expressing aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase. The transgenic plantor cell is genetically modified to knock-out a gene encoding theendogenous flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase thereof tothereby abolish production offlavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in the transgenicplant or cell. One ordinarily skilled in the art can readily design aknock-out construct including both positive and negative selection genesfor efficiently selecting transfected cells that underwent a homologousrecombination event with the construct. Such cells can be cultured andpropagated and can further be induced to differentiate into seedlingsand grown plants, as well known in the art. Further details concerninguses and procedures of plant gene knock-out are found in Miao et al.,1995; Strepp et al., 1998; and Cooley et al., 1998, which areincorporated by reference as if fully set forth herein.

The flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase gene according tothe present invention can be used in a process for enzymaticallymodifying a 1-6 linkage of hesperidin to a 1-2 linkage of neohesperidinby removing and re-coupling the terminal rhamnose. This is done in a twostep process as shown in the left path of FIG. 1. First, hesperidin fromorange peels is treated with immobilized commercial hesperidinase toremove the 1-6 linkage. This process is already used commercially as ade-bittering step for citrus juice. The second step involves using atransgenic organism heavily expressing the gene to re-rhammosylate H7Gin the correct 1-2 position. The organism must be capable of naturallyproducing copious quantities of UDP- or dTDP-rhamnose.

Thus, further according to the present invention, there is provided acommercial process of producing neohesperidin. The process is effectedby implementing the following steps, in which, in a first step,hesperidin is obtained, for example, by extraction from a hesperidinproducing species of the genus Citrus such as orange. Then, thehesperidin is treated with a hesperidinase, therebyhesperetin-7-glucoside is obtained. Thereafter, thehesperetin-7-glucoside is treated with aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in the presence ofactivated rhamnose, such as UDP-rhamnose or dTDP-rhamnose, and therebyneohesperidin is obtained.

Further according to the present invention there is provided acommercial process of producing neohesperidin dihydrochalcone. Theprocess is effected by implementing the following steps, in which, in afirst step, hesperidin is obtained, for example, by extraction from ahesperidin producing species of the genus Citrus such as orange. Then,the hesperidin is treated with a hesperidinase, therebyhesperetin-7-glucoside is obtained. Thereafter, thehesperetin-7-glucoside is treated with aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in the presence ofactivated rhamnose, thereby neohesperidin is obtained. Subsequently, theneohesperidin is treated with an alkali, thereby neohesperidin chalconeis obtained. Finally, the neohesperidin chalcone is reduced, e.g., by aPd catalyzed H₂ surface reduction process, thereby neohesperidindihydrochalcone is obtained.

Preferably, the hesperidinase is immobilized on a solid support, whereasthe hesperidin is treated with the hesperidinase while passing over thesolid support.

Treating the hesperetin-7-glucoside with aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in the presence ofactivated rhamnose is preferably effected in vivo by a cell geneticallymodified to overexpress theflavanone-7-glucoside-2″-O-rhamnosyl-transferase, wherein the cellselected produces activated rhamnose and is capable of intake of thehesperetin-7-glucoside. The neohesperidin in then extracted from thecell. Preferably, the neohesperidin is extracted from the cell prior tothe treatment thereof with the alkali. The cell can be of a bacterium, afungus, a yeast or of a higher plant, as long as it is capable ofgenerating activated rhamnose and is capable of intake of thehesperetin-7-glucoside. The cell can be of a microorganism or of ahigher plant. Suitable microorganisms include bacteria and yeast cells.Lactobacillus casei, Lactobacillus delbrueckeii and Saccharomycescerevisiae provide good examples for microorganisms. Tobacco (Nicotianatabacum), grapes (Vitis viniferis) and carrot (Daucus carota) providegood examples for higher plant cells which are readily propagated inculture and qualify with the required criteria.

This aspect of the present invention is of high significance. It is wellknown that in vitro formation of activated sugar moieties is highlyinefficient. Therefore, only when the supply of activated rhamnose iseffected in vivo by an organism capable also of intake ofhesperetin-7-glucoside, either naturally or rendered capable thereof byphysical, chemical, biological or genetic means, and of overexpressingflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in stoichiometricamounts, would the process described herein be amenable toindustrialization.

Further according to the present invention there is provided a method ofmodifying a rhamnose-1-6-glucose linkage of a chemical compound to arhamnose-1-2-glucose linkage. The method is effected by implementing thefollowing method steps, in which, in a first step, for removal of therhamnose group, the chemical compound is enzymatically treated, therebya first derivative of the chemical compound having a terminal glucosegroup is obtained. Then, the first derivative is enzymatically treatedwith recombinant flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase inthe presence of activated rhamnose, thereby a second derivative chemicalcompound having the rhamnose-1-2-glucose linkage is obtained.Enzymatically treating the first derivative with aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in the presence ofactivated rhamnose is preferably effected in vivo by a cell geneticallymodified to overexpress theflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase, wherein the cellproduces activated rhamnose and is capable of intake of the firstderivative. The second derivative can be extracted from the cell.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Isolation of a Gene Encoding aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase.

Previous attempts to isolate the gene based on purified protein wereunsuccessful. These included cDNA library screens using oligonucleotidesas well as antibodies. The successful approach, RT-PCR using degenerateprimers followed by RACE is described herein.

A cDNA expression library of pomelo young leaves was constructed inLambda-gt111. Antibodies prepared against pomelo 1-2rhamnosyl-transferase were used to screen this library. Positive-lookingclones were later found to be artifacts in that they did not contain arelevant sequence.

Several oligonucleotides were designed according to the partial peptidesequence and were used to screen the above library by standardtechniques. Any resulting clones were irrelevant.

Thus, isolation of the gene encodingflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase (1-2rhamnosyl-transferase) involved a lengthy process initiated in thepurification to homogeneity of the protein, followed by partial peptidesequencing and finalized by cDNA isolation based on RT-PCR and RACE.

Details of the process are as follows:

Purification of the enzyme and activity assay: Purification andfunctional analysis of theflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase protein from youngpomelo leaves was as described (Bar-Peled et al. 1991).

Partial peptide sequence of the protein: The purified protein wassubject to partial proteolysis, using several different proteases(Lys-C; Asp-N; Trypsin), followed by separation of peptides by SDS-PAGEand blotting on PVDF membranes. Partial peptide sequences generated byEdman degradation were as follows (only conclusive sequence data isgiven; amino acids in question are not shown): Lys-C cleavage yieldedtwo partial sequences: 1 N Y F L H L T A (SEQ ID NO:1) and Y P F (SEQ IDNO:2); Asp-N cleavage yielded four partial sequences: I A A I L F L (SEQID NO:3), Y F P S L M G N (SEQ ID NO:4), E K M T I E E A (SEQ ID NO:5)and L F Q P (SEQ ID NO:6); whereas, trypsin cleavage yielded a singlepartial sequence: V V D N G M G M V V P R D K (SEQ ID NO:7).

Isolation of partial cDNA fragments: Partial cDNA fragments wereobtained by an RT-PCR approach on target tissue mRNA (young pomeloleaves) using degenerate primers based on the partial peptide sequences.Pomelo young leaf single strand cDNA was prepared by reversetranscription of poly-A RNA using a PCR-adapted poly-T primer (primercontains sequences identical to the −40M13 forward primer followed by 15T's; see below). PCR was used to amplify gene specific fragments usingdegenerate primers (designed according to partial peptide sequences; seebelow) in conjunction with the −40M13 forward primer. Primer sequenceswere as follows:

Gene specific degenerate primers were: 5′-GAT(C)AAT(C)GGIAT GGGIATGGT-3′(SEQ ID NO:8) for D N G M G M V (SEQ ID NO:9);5′-GAA(G)AAG(A)ATGACIATT(CA)GAA(G)GAA(G)GC-3′ (SEQ ID NO:10) for E K M TI E E A (SEQ ID NO:5); and 5′-AAC(T)TAC(T)TTC(T) CTICAC(T)CTIACIGC-3′(SEQ ID NO:11) for N Y F L H L T A (SEQ ID NO:12).

General primers were: PCR-adapted poly-T primer 5′-GTTTTCCCAGTCACGACGTTTTTTTTTTTTTTT (SEQ ID NO:13); and −40M13 forward primer5′-GTTTTCCCAGTCACGACG-3′ (SEQ ID NO: 14).

Amplified DNA fragments were separated and isolated from agarose gelsand then cloned into pGEM-T vector (Promega, Madison Wis.). Sequence wasobtained by automated sequencing using universal primers (T7 and SP6).

Completion of cDNA sequence: The full length cDNA sequence was obtainedafter isolating the missing 5′ segment by 5′-RACE (Schaefer B. C., 1995)using the following gene specific primers which were derived from thepartial cDNA sequence data: RACE1: 5′-CATGCCCATACCATTGTC-3′ (SEQ IDNO:15); RACE2: 5′-GACAATGGTATGGGCATG-3′ (SEQ ID NO: 16); and RACE3:5′-CCTCAACCACCGAGCCCCAACCAC-3′ (SEQ ID NO: 17).

Based on the total assembled sequence data, the complete coding regionwas isolated by RT-PCR using a proof-reading thermostable polymerase(Pfu; Stratagene, La-Jolla, Calif.) and the following primers (eachprimer contains a cloning adapter sequence; gene specific sequences areunderlined): 5′-CATCTAGAATGGATACCAAGCATCAAG-3′ (SEQ ID NO: 18); and5′-CAGGATCCTTATTCAGATTTCTTGACAAG-3′ (SEQ ID NO:19).

The resulting translated coding region (1359 base-pairs; FIG. 2, SEQ IDNO: 20) contains all partial peptide sequences previously determined byprotein sequencing. The sequence of the protein encoded by SEQ ID NO: 20is given in SEQ ID NO:21.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

REFERENCES CITED

-   1. Barber, G. A. (1962) The enzyme synthesis of uridine disphosphate    L-rhamnose. Biochem. Biophys. Res. Commun 8: 204-209.-   2. Bar-Peled, M., E. Lewinsohn, R. Fluhr and J. Gressel. 1991.    UDP-rhanmose: flavanone-7-O-glucoside-2-O-rhamnosyl-transferase:    purification and characterization of an enzyme catalyzing the    production of bitter compounds in citrus. J. Biol. Chem. 266:    20953-20959.-   3. Berhow, M. A., and Vandercook C. E. (1989) Biosynthesis of    naringin and prunin in detached grapefruit. Phytochemistry 28:    1627-1630.-   4. Castillo, J., O. Benavente and J. A. del R10. (1992) Naringin and    neohesperidin levels during development of leaves, flower buds and    fruits of Citrus aurantium. Plant Physiol. 99: 67-73.-   5. Castillo, J., O. Benavente and J. A del R10 (1993) Hesperetin    7-O-Glucoside and prunin in Citrus species (C. aurantium and C.    paradisi). A study of their quantitative distribution in immature    fruits and as immediate precursors of neohesperidin and naringin    in C. aurantium. J. Agric. Food Chem. 41: 1920-1924.-   6. Cooley M. B., Yoder J. L., Insertional inactivation of the tomato    polygalacteronase gene. Plant Mol. Biol. 1998, 38: 521-530.-   7. Guadagni, D. G. V. P. Maier and J. G. Turnbaugh (1973) Effect of    some citrus juice constituents on taste thresholds for limonin and    naringin bitterness. J. Sci. Fd Agric. 24: 1277-1288.-   8. Horowitz, R. M. and Gentili, B. (1977) Flavonoid constituents of    citrus in “Citrus Science and Technology”, S. Nagy et al., eds. AVI    Publishing Co., Westport, Conn. Vol. 1, pp. 397-426.-   9. Horowitz, R. M. and Gentili, B. (1986) Dihydrochalcone sweeteners    from citrus flavanones in “Alternative Sweetners”, L. O. Nabors    and R. C. Gelardi, eds. Marcel Dekker, Inc., NY. pp. 135-153.-   10. Jimeno, A., Manjon, A. Canovas, M. and Iborra, J. L. (1987) Use    of naringinase immobilized on glycophase-coated porous glass for    fruit juice debittering. 13-16.-   11. Lewinsohn, E., E. Berman, Y. Mazur and J. Gressel. 1986.    Glucosylation of exogenous flavanones by grapefruit (Citrus    paradisi) cell cultures. Phytochemistry 25:2531-2535.-   12. Lewinsohn, E., E. Berman, Y. Mazur and J. Gressel. 1989a.    (7)Glucosylation and (1-6)rhamnosylation of exogenous flavanones by    undifferentiated Citrus cell cultures. Plant Science 61:23-28.-   13. Lewinsohn, E., L. Britsch, Y. Mazur and J. Gressel. 1989b.    Flavanone glycoside biosynthesis in Citrus: chalcone synthase,    UDP-glucose: flavanone-7-O-glucosyl-transferase and rhamnosyl    transferase activities in cell-free extracts. Plant Physiology    91:1323-1328.-   14. Matthews, R. F., Rouseff, R. L., Manlan, M., Norman, S. I. 1990.    Removal of limonin and naringin from citrus juice by styrene    divinylbenzene resins. Food. Tech. (April) 130-132.-   15. McIntosh, C. A. and Mansell, R. L. (1990) Biosynthesis of    naringin in Citrus paradisi: UDP-glucosyl-transferase activity in    grapefruit seedlings. Phytochemistry, 29: 1533-1538.-   16. Miao Z. H., Lam E. Targeted disruption of the TGA3 locus in    arabidopsis thaliana. Plant J. 1995, 7:359-365.-   17. Naim, M., E. Dukan, U. Zehavi and L. Yaron. (1986) The    water-sweet aftertaste of neohesperidin dihydrochalcone and    thaumatin as a method for determining their sweet persistence. Chem.    Senses. 11: 361-370.-   18. Schaefer B. C. Revolutions in rapid amplification of cDNA ends:    New strategies for polymerase chain reaction cloning of full-length    cDNA ends. Analytical Biochemistry, 1995, 227:255-273.-   19. Strepp R., Scholz S., Kruse S., Speth V., Reski R. Plant nuclear    gene knockout reveals a role in plastid division for the homologue    of the bacterial cell division protein FtsZ, an ancestral tubulin.    Proc. Natl. Acad. Sci USA 1998, 95: 4368-4373.

1-47. (canceled)
 48. A commercial process of producing neohesperidincomprising the steps of: (a) obtaining hesperidin; (b) treating saidhesperidin with a hesperidinase, thereby obtaininghesperetin-7-glucoside; and (c) treating said hesperetin-7-glucosidewith a flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in thepresence of activated rhamnose, thereby obtaining neohesperidin.
 49. Thecommercial process of claim 48, wherein said activated rhamnose isselected from the group consisting of UDP-rhamnose or dTDP-rhamnose. 50.The commercial process of claim 48, wherein said hesperidinase isimmobilized on a solid support, whereas said hesperidin is treated withsaid hesperidinase while passing over said solid support.
 51. Thecommercial process of claim 48, wherein said treatment ofhesperetin-7-glucoside with aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in presence ofactivated rhamnose of step (c) is effected in vivo by a cell geneticallymodified to overexpress saidflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase, which said cell isproducing activated rhamnose and is capable of intake of saidhesperetin-7-glucoside.
 52. The commercial process of claim 51, furthercomprising the step of extracting said neohesperidin from said cell. 53.A commercial process of producing neohesperidin dihydrochalconecomprising the steps of: (a) obtaining hesperidin; (b) treating saidhesperidin with a hesperidinase, thereby obtaininghesperetin-7-glucoside; (c) treating said hesperetin-7-glucoside with aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in the presence ofactivated rhamnose, thereby obtaining neohesperidin; (d) treating saidneohesperidin with an alkali, thereby obtaining neohesperidin chalcone;and (e) reducing said neohesperidin chalcone, thereby obtainingneohesperidin dihydrochalcone
 54. The commercial process of claim 53,wherein said activated rhamnose is selected from the group consisting ofUDP-rhamnose or dTDP-rhamnose.
 55. The commercial process of claim 53,wherein said hesperidinase is immobilized on a solid support, whereassaid hesperidin is treated with said hesperidinase while passing oversaid solid support.
 56. The commercial process of claim 53, wherein saidtreatment of hesperetin-7-glucoside with aflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase in presence ofactivated rhamnose of step (c) is effected in vivo by a cell geneticallymodified to overexpress saidflavanone-7-O-glucoside-2″-O-rhamnosyl-transferase, which said cell isproducing activated rhamnose and is capable of intake of saidhesperetin-7-glucoside.
 57. The commercial process of claim 56, furthercomprising the step of extracting said neohesperidin from said cellprior to treating said neohesperidin with said alkali. 58.-83.(canceled)